Reference is also made to the applications of Audonnet et al., Ser. Nos. 09/232,278, 09/232,479, 09/232,477, 09/232,279, and 09/232,478 and to the application of Rijsewijk et al. Ser. No. 09/232,469, all filed Jan. 15, 1999. All of the above-mentioned applications, as well as all documents cited herein and documents referenced or cited in documents cited herein, are hereby incorporated herein by reference. Vectors of vaccines or immunological compositions of the aforementioned applications, as well as of documents cited herein or documents referenced or cited in documents cited herein or portions of such vectors (e.g., one or more or all of regulatory sequences such as DNA for promoter, leader for secretion, terminator), may to the extent practicable with respect to the preferred host of this application, also be employed in the practice of this invention; and, DNA for vectors of vaccines or immunological compositions herein can be obtained from available sources and knowledge in the art, e.g., GeneBank, such that from this disclosure, no undue experimentation is required to make or use such vectors.
The present invention relates to a vaccine formula allowing in particular the vaccination of pigs against reproductive and respiratory pathologies. It also relates to a corresponding method of vaccination.
During the past decades, the methods for the production of pigs have changed fundamentally. The intensive breeding in an enclosed space has become generalized with, as a corollary, the dramatic development of respiratory pathologies.
The range of symptoms of porcine respiratory pathology is in general grouped under the complex name of pig respiratory disease and involves a wide variety of pathogenic agents comprising viruses as well as bacteria and mycoplasmas.
The principal agents involved in the respiratory disorders are Actinobacillus pleuropneumoniae, the infertility and respiratory syndrome virus (PRRS) also called mysterious disease virus, the Aujeszky""s disease virus (PRV) and the swine flu virus.
Other viruses cause reproductive disorders leading to abortions, mummifications of the foetus and infertility. The principal viruses are PRRS, the parvovirus and the conventional hog cholera virus (HCV). Secondarily, the swine flu virus PRV and A. pleuropneumoniae can also cause such disorders. Deaths may occur with A. pleuropneumoniae, HCV and PRV.
In addition, interactions between microorganisms are very important in the porcine respiratory complex. Indeed, most of the bacterial pathogens are habitual hosts of the nasopharangeal zones and of the tonsils in young animals. These pathogens, which are derived from the sows, are often inhaled by the young pigs during their first few hours of life, before the cholostral immunity has become effective. The organisms living in the upper respiratory tract may invade the lower tract when the respiratory defense mechanisms of the host are damaged by a precursor agent such as A. pleuropneumoniae or by viruses. The pulmonary invasion may be very rapid, in particular in the case of precursor pathogens such as A. pleuropneumoniae which produce potent cytotoxins capable of damaging the cilia of the respiratory epithelial cells and the alveolar macrophages.
Major viral infections, such as influenza, and respiratory coronavirus and Aujeszky""s virus infections, may play a role in the pathogenicity of the respiratory complex, besides bacteria with respiratory tropism and mycoplasmas.
Finally, some agents have both a respiratory and a reproductive effect. Interactions may also occur from the point of view of the pathology of reproduction.
It therefore appears to be necessary to try to develop an effective prevention against the principal pathogenic agents involved in porcine reproductive and respiratory pathologies.
The associations developed so far were prepared from inactivated vaccines or live vaccines and, optionally, mixtures of such vaccines. Their development poses problems of compatibility between valencies and of stability. It is indeed necessary to ensure both the compatibility between the different vaccine valencies, whether from the point of view of the different antigens used from the point of view of the formulations themselves, especially in the case where both inactivated vaccines and live vaccines are combined. The problem of the conservation of such combined vaccines and also of their safety especially in the presence of an adjuvant also exists. These vaccines are in general quite expensive.
Patent applications WO-A-90 11092, WO-A-93 19183, WO-A-94 21797 and WO-A-95 20660 have made use of the recently developed technique of polynucleotide vaccines. It is known that these vaccines use a plasmid capable of expressing, in the host cells, the antigen inserted into the plasmid. All the routes of administration have been proposed (intraperitoneal, intravenous, intramuscular, transcutaneous, intradermal, mucosal and the like). Various vaccination means can also be used, such as DNA deposited at the surface of gold particles and projected so as to penetrate into the animals"" skin (Tang et al., Nature, 356, 152-154, 1992) and liquid jet injectors which make it possible to transfect at the same time the skin, the muscle, the fatty tissues and the mammary tissues (Furth et al., Analytical Biochemistry, 205, 365-368, 1992). (See also U.S. Pat. Nos. 5,846,946, 5,620,896, 5,643,578, 5,580,589, 5,589,466, 5,693,622, and 5,703,055; Science, 259:1745-49, 1993; Robinson et al., seminars in IMMUNOLOGY, 9:271-83, 1997; Luke et al., J. Infect. Dis. 175(1):91-97, 1997; Norman et al., Vaccine, 15(8):801-803, 1997; Bourne et al., The Journal of Infectious Disease, 173:800-7, 1996; and, note that generally a plasmid for a vaccine or immunological composition can comprise DNA encoding an antigen operatively linked to regulatory sequences which control expression or expression and secretion of the antigen from a host cell, e.g., a mammalian cell; for instance, from upstream to downstream, DNA for a promoter, DNA for a eukaryotic leader peptide for secretion, DNA for the antigen, and DNA encoding a terminator.).
The polynucleotide vaccines may also use both naked DNAs and DNAs formulated, for example, inside cationic lipid liposomes.
M-F Le Potier et al., (Second International Symposium on the Eradication of Aujeszky""s Disease (pseudorabies) Virus Aug. 6th to 8th 1995 Copenhagen, Denmark) and M. Monteil et al., (Les Journxc3xa9es d""Animation Scientifique du Dxc3xa9partement de Pathologie Animale [Scientific meeting organized by the department of animal pathology], INRA-ENV, Ecole Nationale Vxc3xa9txc3xa9rinaire, LYON, Dec. 13-14, 1994) have tried to vaccinate pigs against the Aujeszky""s disease virus with the aid of a plasmid allowing the expression of the gD gene under the control of a strong promoter, the type 2 adenovirus major late promoter. In spite of a good antibody response level, no protection could be detected. Now, satisfactory results in the area of protection have been recorded after inoculation of pigs with a recombinant adenovirus into which the gD gene and the same promoter have been inserted, proving that the gD glcyoprotein could be sufficient for inducing protection in pigs.
The prior art gives no protective result in pigs by the polynucleotide vaccination method.
The invention proposes to provide a multivalent vaccine formula which makes it possible to ensure vaccination of pigs against a number of pathogenic agents involved in particular in respiratory pathology and/or in reproductive pathology.
Another objective of the invention is to provide such a vaccine formula combining different valencies while exhibiting all the criteria required for mutual compatibility and stability of the valencies.
Another objective of the invention is to provide such a vaccine formula which makes it possible to combine different valencies in the same vehicle.
Another objective of the invention is to provide such a vaccine formula which is easy and inexpensive to use.
Yet another objective of the invention is to provide such a vaccine formula and a method for vaccinating pigs which makes it possible to obtain protection, including multivalent protection, with a high level of efficiency and of long duration, as well as good safety and an absence of residues.
The subject of the present invention is therefore a vaccine formula in particular against porcine reproductive and/or respiratory pathology, comprising at least 3 polynucleotide vaccine valencies each comprising a plasmid integrating, so as to express it in vivo in the host cells, a gene with one porcine pathogen valency, these valencies being selected from those of the group consisting of Aujeszky""s disease virus (PRV or pseudorabies virus), swine flu virus (swine influenza virus, SIV), pig mysterious disease virus (PRRS virus), parvovirosis virus (PPV virus), conventional hog cholera virus (HCV virus) and bacterium responsible for actinobacillosis (A. pleuropneumoniae), the plasmids comprising, for each valency, one or more of the genes selected from the group consisting of gB and gD for the Aujeszky""s disease virus, HA, NP and N for the swine flu virus, ORF5 (E), ORF3, ORF6 (M) for the PRRS virus, VP2 for the parvovirosis virus, E1, E2 for the conventional hog cholera virus and apxI, apxII and apxIII for A. pleuropneumoniae. 
Valency in the present invention is understood to mean at least one antigen providing protection against the virus for the pathogen considered, it being possible for the valency to contain, as subvalency, one or more modified natural genes from one or more strains of the pathogen considered.
Pathogenic agent gene is understood to mean not only the complete gene but also the various nucleotide sequences, including fragments which retain the capacity to induce a protective response. The notion of a gene covers the nucleotide sequences equivalent to those described precisely in the examples, that is to say the sequences which are different but which encode the same protein. It also covers the nucleotide sequences of other strains of the pathogen considered, which provide cross-protection or a protection specific for a strain or for a strain group. It also covers the nucleotide sequences which have been modified in order to facilitate the in vivo expression by the host animal but encoding the same protein.
Preferably, the vaccine formula according to the invention will comprise the Aujeszky and porcine flu valencies to which other valencies, preferably selected from the PRRS and A. pleuropneumoniae (actinobacillosis) valencies, can be added. Other valencies selected from the parvovirosis and conventional hog cholera valencies can be optionally added to them.
It goes without saying that all the combinations of valencies are possible. However, within the framework of the invention, the Aujeszky and porcine flu, followed by PRRS and A. pleuropneumoniae, valencies are considered to be preferred.
From the point of viewing of a vaccination directed more specifically against the porcine respiratory pathology the valencies will be preferably selected from Aujeszky, porcine flu, PRRS and actinobacilosis.
From the point of view of a vaccination directed specifically against the reproductive pathology, the valencies will be preferably selected from PRRS, parvovirosis, conventional hog cholera and Aujeszky.
As regards the Aujeszky valency, either of the gB and gD genes may be used. Preferably, both genes are used, these being in this case mounted in different plasmids or in one and the same plasmid.
As regards the porcine flu valency, the HA and NP genes are preferably used. Either of these two genes or both genes simultaneously can be used, mounted in different plasmids or in one and the same plasmid. Preferably, the HA sequences from more than one influenza virus strain, in particular from the different strains found in the field, will be combined in the same vaccine. On the other hand, NP provides cross-protection and the sequence from a single virus strain will therefore be satisfactory.
As regards the PRSS valency, the E and ORF3 or alternatively M genes are preferably used. These genes can be used alone or in combination; in the case of a combination, the genes can be mounted into separate plasmids or into plasmids combining 2 or 3 of these genes. Genes derived from at least two strains, especially from a European strain and an American strain, will be advantageously combined in the same vaccine.
As regards the conventional hog cholera valency, either of the E1 and E2 genes or also E1 and E2 genes combined, in two different plasmids or optionally in one and the same plasmid, can be used.
As regards the actinobacillosis valency, one of the three genes mentioned above or a combination of 2 or 3 of these genes, mounted in different plasmids or mixed plasmids, may be used in order to provide protection against the different serotypes of A. pleuropneumoniae. For the apxI, II and III antigens, it may be envisaged that the coding sequences be modified in order to obtain the detoxified antigens, in particular as in the examples.
The vaccine formula according to the invention can be provided in the form of a dose volume generally of between 0.1 and 10 ml, and in particular between 1 and 5 ml especially for vaccinations by the intramuscular route.
The dose will be generally between 10 ng and 1 mg, preferably between 100 ng and 50 xcexcg and preferably between 1 xcexcg and 250 xcexcg per plasmid type.
Use will preferably be made of naked plasmids simply placed in the vaccination vehicle which will be in general physiological saline (0.9% NaCl), ultrapure water, TE buffer and the like. All the polynucleotide vaccine forms described in the prior art can of course be used.
Each plasmid comprises a promoter capable of ensuring the expression of the gene inserted, under its control, into the host cells. This will be in general a strong eukaryotic promoter and in particular a cytomegalovirus early CMV-IE promoter of human or murine origin, or optionally of another origin such as rats, pigs and guinea pigs.
More generally, the promoter may be either of viral origin or of cellular origin. As viral promoter, there may be mentioned the SV40 virus early or late promoter or the Rous sarcoma virus LTR promoter. It may also be a promoter from the virus from which the gene is derived, for example the gene""s own promoter.
As cellular promoter, there may be mentioned the promoter of a cytoskeleton gene, for example the desmin promoter (Belmont et al., Journal of Submicroscopic Cytology and Pathology, 1990, 22, 117-122; and Zhenlin et al., Gene, 1939, 78, 243-254), or alternatively the actin promoter.
When several genes are present in the same plasmid, these may be presented in the same transcription unit or in two different units.
The combination of the different vaccine valencies according to the invention may be preferably achieved by mixing the polynucleotide plasmids expressing the antigen(s) of each valency, but it is also possible to envisage causing antigens of several valencies to be expressed by the same plasmid.
The subject of the invention is also monovalent vaccine formulae comprising one or more plasmids encoding one or more genes from one of the viruses selected from the group consisting of PRV, PRRS, PPV, HCV and A. pleuropneumoniae, the genes being those described above. Besides their monovalent character, these formulae may possess the characteristics stated above as regards the choice of the genes, their combinations, the composition of the plasmids, the dose volumes, the doses and the like.
The monovalent vaccine formulae may be used (i) for the preparation of a polyvalent vaccine formula as described above, (ii) individually against the actual pathology, (iii) combined with a vaccine of another type (live or inactivated whole, recombinant, subunit) against another pathology, or (iv) as booster for a vaccine as described below.
The subject of the present invention is in fact also the use of one or more plasmids according to the invention for the manufacture of a vaccine intended to vaccinate pigs first vaccinated by means of a first conventional vaccine of the type in the prior art, namely, in particular, selected from the group consisting of a live whole vaccine, an inactivated whole vaccine, a subunit vaccine, a recombinant vaccine, this first vaccine (monovalent or multivalent) having (that is to say containing or capable of expressing) the antigen(s) encoded by the plasmids or antigen(s) providing cross-protection. Remarkably, the polynucleotide vaccine has a potent booster effect which results in an amplification of the immune response and the acquisition of a long-lasting immunity.
In general, the first-vaccination vaccines can be selected from commercial vaccines available from various veterinary vaccine producers.
The subject of the invention is also a vaccination kit grouping together a first-vaccination vaccine as described above and a vaccine formula according to the invention for the booster. It also relates to a vaccine formula according to the invention accompanied by a leaflet indicating the use of this formula as a booster for a first vaccination as described above.
The subject of the present invention is also a method for vaccinating pigs against the porcine reproductive pathology and/or respiratory pathology, comprising the administration of an effective dose of a vaccine formula as described above. This vaccination method comprises the administration of one or more doses of the vaccine formula, it being possible for these doses to be administered in succession over a short period of time and/or in succession at widely spaced intervals.
The vaccine formulae according to the invention can be administered in the context of this method of vaccination, by the different routes of administration proposed in the prior art for polynucleotide vaccination and by means of known techniques of administration. The vaccination can in particular be used by the intradermal route with the aid of a liquid jet, preferably multiple jet, injector and in particular an injector using an injection head provided with several holes or nozzles, in particular comprising from 5 or 6 holes or nozzles, such as the Pigjet apparatus manufactured and distributed by the company Endoscoptic, Laons, France.
The dose volume for such an apparatus will be reduced preferably to between 0.1 and 0.9 ml, in particular between 0.2 and 0.6 ml and advantageously between 0.4 and 0.5 ml, it being possible for the volume to be applied in one or several, preferably 2, applications.
The subject of the invention is also the method of vaccination consisting in making a first vaccination as described above and a booster with a vaccine formula according to the invention. In a preferred embodiment of the process according to the invention, there is administered in a first instance, to the animal, an effective dose of the vaccine of the conventional, especially inactivated, live, attenuated or recombinant, type, or alternatively a subunit vaccine, so as to provide a first vaccination, and, after a period preferably of 2 to 6 weeks, the polyvalent or monovalent vaccine according to the invention is administered.
The invention also relates to the method of preparing the vaccine formulae, namely the preparation of the valencies and mixtures thereof, as evident from this description.
The invention will now be described in greater detail with the aid of the embodiments of the invention taken with reference to the accompanying drawings.